Pathogenesis of mitochondrial dysfunction in skeletal muscle

Abstract

Mitochondrial diseases are amongst the most prevalent genetic disorders, however little is known about genetic and cellular mechanisms behind disease pathogenesis and progression. Elucidating such mechanisms can help identify targets for novel therapeutic measures and improve patient care by informing the implementation of clinical regimens and providing clearer information on prognoses. This project aims to improve the understanding of the molecular mechanisms behind the pathogenesis of mitochondrial dysfunction in muscle and the genetic and biochemical changes occurring over time in patients with mitochondrial disease. Firstly, a longitudinal study combines immunofluorescent and molecular genetic techniques to assess biochemical and genetic changes over time in serial skeletal muscle biopsies from patients with m.3243A>G or single, large-scale mtDNA deletions, the two largest groups in the MRC Centre Mitochondrial Disease Patient Cohort. Further investigation into the relationship between the genetic and biochemical defects in patients with single, large-scale mtDNA deletions is carried out by applying a single-fibre approach. Here, muscle fibres are classified by their biochemical defect and laser microdissected for genetic analysis to determine deletion level and mtDNA copy number. These studies find that: (i) changes to mutation level, mtDNA copy number and biochemical defect occur over time in skeletal muscle of mitochondrial disease patients; (ii) these changes are inconsistent in magnitude and direction across groups of patients and (iii) the biochemical threshold for deficiency is affected by the size and location of single, large-scale mtDNA deletions. In addition, a real time PCR assay for the quantification of mitochondrial DNA copy number from homogenate tissue has been optimised to improve accuracy through the use of additional gene markers.